Novel peptides

ABSTRACT

The invention relates to an isolated, synthetic or recombinant χ-conotoxin peptide having the ability to inhibit a neuronal amine transporter, nucleic acid molecules encoding all or part of such peptides, antibodies to such peptides and uses and methods of treatment involving them.

The present invention relates to novel peptides and derivatives thereofuseful as inhibitors of neuronal amine transporters of neurotransmitterssuch as noradrenaline, serotonin, dopamine, glutamic acid and glycine.The invention also relates to pharmaceutical compositions comprisingthese peptides, nucleic acid probes useful in finding active analoguesof these peptides, assays for finding compounds having neuronalnoradrenaline transporter inhibitory activity and the use of thesepeptides in the prophylaxis or treatment of conditions such as but notlimited to incontinence, cardiovascular conditions and mood disorders.

The marine snails of the genus Conus (cone snails) use a sophisticatedbiochemical strategy to capture their prey. As predators of either fish,worms or other molluscs, the cone snails inject their prey with venomcontaining a cocktail of small bioactive peptides. These toxinmolecules, which are referred to as conotoxins, interfere withneurotransmission by targeting a variety of receptors and ion-channels.The venom from any single Conus species may contain more than 100different peptides. The conotoxins are divided into classes on the basisof their physiological targets. To date, ten classes have beendescribed. The ω-conotoxin class of peptides target and blockvoltage-sensitive Ca²⁺-channels inhibiting neurotransmitter release. Theα-conotoxins and ψ-conotoxins target and block nicotinic ACh receptors,causing ganglionic and neuromuscular blockade. Peptides of theμ-conotoxin class act to block voltage-sensitive Na⁺-channels inhibitingmuscle and nerve action potentials. The δ-conotoxins target and delaythe inactivation of voltage-sensitive Na⁺-channels, enhancing neuronalexcitability. The κ-conotoxin class of peptides target and blockvoltage-sensitive K⁺-channels, and these also cause enhanced neuronalexcitability. The conopressins are vasopressin receptor antagonists andthe conantokins are NMDA receptor antagonists. More recently, theprototype of a new γ-conotoxin class, which targets a voltage-sensitivenonspecific cation channel, and of a new σ-conotoxin class, whichantagonises the 5HT₃ receptor, have been described.

It has now been found that a new class of conotoxin exists, hereinafterreferred to as the χ-conotoxin class, which are characterised by havingthe ability to inhibit neuronal amine transporters.

Compounds which inhibit neurotransmitter reuptake have been found to beuseful in the treatment of lower urinary tract disorders, such asurinary incontinence, detrusor instability and interstitial cystitis.One such compound is “imipramine” which, in addition to inhibitingnoradrenaline reuptake, has been shown to affect calcium channelblockade, and to exhibit anticholinergic, local anaesthetic activity anda number of other effects. Other compounds capable of inhibitingnoradrenaline reuptake are described in U.S. Pat. No. 5,441,985. Thesecompounds are said to have a reduced anticholinergic effect relative toimipramine.

In the case of the peptides of the present invention this inhibition ofneurotransmitter reuptake is achieved by selectively inhibiting theneuronal neurotransmitter transporter, such as the noradrenalinetransporter, which functions to rapidly clear released noradrenalinefrom the synapse back into neurons.

The peptides of the present invention are the first peptides to haveactivity in inhibiting an amine transporter. All other conotoxinpeptides characterised to date target ion channels or receptors on cellsurfaces.

According to one aspect of the present invention there is provided anisolated, synthetic or recombinant χ-conotoxin peptide having theability to inhibit a neuronal amine transporter.

Preferably, the neuronal amine transporter is the neuronal noradrenalinetransporter.

The χ-conotoxin peptide may be a naturally occurring peptide isolatedfrom a cone snail, or a derivative thereof.

Preferably the χ-conotoxin peptide is χ-MrIA or χ-MrIB, or a derivativethereof. χ-MrIA and χ-MrIB may be isolated from the venom of the molluschunting cone snail, Conus marmoreus.

They are both peptides of 13 amino acid residues in length, and contain2-disulphide bonds; the peptides show most homology to members in theα-conotoxin class, which act as nicotinic ACh receptor antagonists.

The amino acid sequences of χ-MrIA and χ-MrIB are as follows: χ-MrIANGVCCGYKLCHOC SEQ ID NO. 1 χ-MrIB VGVCCGYKLCHOC SEQ ID NO. 2

The C-terminus may be a free acid or amidated.

In the sequences above the “O” refers to 4-hydroxy proline (Hyp). Thisamino acid residue results from post translational modification of theencoded peptide and is not directly encoded by the nucleotide sequence.

Preferably, the χ-conotoxin peptide is a selective inhibitor of theneuronal noradrenaline transporter. The terms “selective” and“selectively” as used herein mean that the activity of the peptide as aninhibitor of neuronal noradrenaline transporter is considerably greaterthan any activity at the α₁-adrenoceptors.

U.S. Pat. No. 5,441,985 indicates that inhibitors of noradrenalinereuptake which have a negligible anticholinergic effect are particularlyuseful in the treatment of lower urinary tract disorders. It has beenfound that χ-MrIA also has no detectable anticholinergic effect.

Accordingly in a preferred embodiment of the invention there is providedan isolated, synthetic or recombinant χ-conotoxin peptide having theability to selectively inhibit neuronal noradrenaline transporter, andhaving negligible or no anticholinergic effect. χ-MrIA has also beenfound to have no activity as a sodium channel blocker or as an inhibitorof dopamine transporter. The absence in χ-MrIA of these additionalpharmacological activities commonly associated with other noradrenalinetransporter inhibitors and in preferred peptides according to theinvention, makes these peptides useful pharmacological tools.

The χ-conotoxin peptides according to the invention may be naturallyoccurring peptides, such as χ-MrIA and χ-MrIB, or may be derivatives ofnaturally occurring peptides.

The term “derivative” as used herein in connection with naturallyoccurring χ-conotoxin peptides, such as χ-MrIA and χ-MrIB, refers to apeptide which differs from the naturally occurring peptides by one ormore amino acid deletions, additions, substitutions, or side-chainmodifications. Such derivatives which do not have the ability to inhibitneuronal noradrenaline transporter do not fall within the scope of thepresent invention.

Substitutions encompass amino acid alterations in which an amino acid isreplaced with a different naturally-occurring or a non-conventionalamino acid residue. Such substitutions may be classified as“conservative”, in which case an amino acid residue contained in apolypeptide is replaced with another naturally-occurring amino acid ofsimilar character either in relation to polarity, side chainfunctionality or size, for example Ser

Thr

Pro

Hyp

Gly

Ala, Val

Ile

Leu, His

Lys

Arg, Asn

Gln

Asp

Glu or Phe

Trp

Tyr. It is to be understood that some non-conventional amino acids mayalso be suitable replacements for the naturally occurring amino acids.For example ornithine, homoarginine and dimethyllysine are related toHis, Arg and Lys.

Substitutions encompassed by the present invention may also be“non-conservative”, in which an amino acid residue which is present in apeptide is substituted with an amino acid having different properties,such as naturally-occurring amino acid from a different group (eg.substituting a charged or hydrophobic amino acid with alanine), oralternatively, in which a naturally-occurring amino acid is substitutedwith a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be ofmultiple residues, either clustered or dispersed.

Preferably, amino acid substitutions are conservative.

Additions encompass the addition of one or more naturally occurring ornon-conventional amino acid residues. Deletion encompasses the deletionof one or more amino acid residues.

As stated above the present invention includes peptides in which one ormore of the amino acids has undergone sidechain modifications. Examplesof side chain modifications contemplated by the present inventioninclude modifications of amino groups such as by reductive alkylation byreaction with an aldehyde followed by reduction with NaBH₄; amidinationwith methylacetimidate; acylation with acetic anhydride; carbamoylationof amino groups with cyanate; trinitrobenzylation of amino groups with2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groupswith succinic anhydride and tetrahydrophthalic anhydride; andpyridoxylation of lysine with pyridoxal-5-phosphate followed byreduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH. Any modificationof cysteine residues must not affect the ability of the peptide to formthe necessary disulphide bonds. It is also possible to replace thesulphydryl groups of cysteine with selenium equivalents such that thepeptide forms a diselenium bond in place of one or more of thedisulphide bonds.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Proline residue may be modified by, for example, hydroxylation in the4-position.

A list of some amino acids having modified side chains and otherunnatural amino acids is shown in Table 1. TABLE 1 Non-conventionalNon-conventional amino acid Code amino acid Code α-aminobutyric acid AbuL-N-methylalanine Nmala α-amino-α-methylbutyrate MgabuL-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagineNmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethylglycinecarbamylmethylglycine 1-carboxy-1-(2,2- Nmbc O-methyl-L-serine Omserdiphenylethylamino)cyclopropane O-methyl-L-homoserine Omhser

These types of modifications may be important to stabilise the peptideif administered to an individual or used as a diagnostic reagent.

Other derivatives contemplated by the present invention include a rangeof glycosylation variants from a completely unglycosylated molecule to amodified glycosylated molecule. Altered glycosylation patterns mayresult from expression of recombinant molecules in different host cells.

The χ-conotoxins of the present invention are typically amidated at theC-terminal, however compounds with a free carboxyl terminus or othermodifications at the C-terminal are considered to be within the scope ofthe present invention. Preferably the peptides are amidated or have afree carboxyl at the C-terminal.

Preferably the derivatives of naturally occurring χ-conotoxin peptideswill retain the Cys residues and characteristic disulphide bondingpattern. Derivatives may include additional Cys residues provided theyare protected during formation of the disulphide bonds.

In modification to form derivatives of naturally occurring χ-conotoxinpeptides it is useful to compare the amino acid sequences of activenaturally occurring peptides to determine which, if any, of the residuesare conserved between active species. Substitution of these conservedresidues, while not prohibited, is less favoured than substitutions ofnon-conserved residues.

Derivatives where Ala replaces one or more residues can be used toidentify the pharmacophore. Preferably only one or two amino acids isreplaced with Ala at a time. Additional new peptides can be made wherecharged, polar or hydrophobic residues, respectively, are replaced toassist defining more precisely the type of interactions involved in thebinding of this pharmacological class of peptide to its receptor.Non-conservative replacements, where charge is reversed, or polarresidues replace hydrophobic residues, can further identify residuesinvolved in binding. All of these peptides have potential to showimproved potency, or greater selectivity. Non-native amino acid changescould also be included to improve potency, selectivity and/or stability.

Exposed residues are most likely to be involved in receptor binding andcan be systematically replaced. Particular emphasis is placed onchanging residues involved in binding and residues just on the peripheryof the pharmacophore, using longer side chain forms or non-conservedchanges to pick up additional binding interactions for improved potencyand/or selectivity. Reducing or enlarging loop sizes and the tail ofMrIA or MrIB further modifies activity.

It is noted that MrIA and MrIB are composed of a tail (residues 1-3),and two loops (residues 6-9 and 11-12), however the χ-conotoxinpeptides, and derivatives of the present invention are not restricted tothose having this particular arrangement of amino acids and disulphidebonds. Other arrangements are also possible, and provided the resultantpeptide has the requisite activity, a peptide will fall within the scopeof the present invention. Preferably the peptides will have at least twocysteine residues and at least one disulphide bond, or more preferablyfour cysteine residues and two disulphide bonds.

The connectivity of the disulfide bonds in these peptides may beA-B/C-D, A-C/B-D or A-D/B-C, the latter being preferred for MrIA andMrIB. A, B, C and D refer to the first, second, third and fourth Cysresidues involved in disulphide bond formation, respectively.

These peptides can also be labelled and used to establish binding assaysto identify new molecules that act at the same site. For example,labelled ligand of MrIA or MrIB could have tritium included or may haveradio-active iodine or similar attached through a Tyr or otherappropriate residue. A Tyr scan through each peptide will establish asuitable location for incorporation of the Tyr. The inhibition ofbinding of such labelled peptides to tissue homogenates or expressedtransporters by compounds or mixtures would permit identification of newpeptides active at this site, including peptides present in serum andnerve and muscle tissue of mammals, including human tissues. The assaywill also allow identification of non-peptide molecules that also act atthe same site as MrIA and MrIB, and that may have utility as orallyactive forms of these peptides. Labelled peptides will additionallypermit autoradiographic studies to identify the location of the peptidebinding across various tissues.

Portions of these sequences can be used to search ESTR data bases toidentify in mammals peptides or proteins that contain related sequenceinformation that could be used to identify endogenous ligands that actin a similar manner in mammals.

The χ-conotoxins of the present invention may be prepared using standardpeptide synthetic methods followed by oxidative disulfide bondformation. For example, the linear peptides may be synthesised by solidphase methodology using BOC chemistry, as described by Schnoltzer et al(1992). Following deprotection and cleavage from the solid support thereduced peptides are purified using preparative chromatography. Thepurified reduced peptides are oxidised in buffered systems, for exampleas described in example 2. The oxidised peptides were purified usingpreparative chromatography.

References describing the synthesis of conotoxins include Sato et al,Lew et al and WO 91/07980.

The χ-conotoxins may also be prepared using recombinant DNA technology.A nucleotide sequence encoding the desired peptide sequence may beinserted into a suitable vector and protein expressed in an appropriateexpression system. In some instances, further chemical modification ofthe expressed peptide may be appropriate, for example C-terminalamidation. Under some circumstances it may be desirable to undertakeoxidative bond formation of the expressed peptide as a chemical stepfollowing peptide expression. This may be preceded by a reductive stepto provide the unfolded peptide. Those skilled in the art may readilydetermine appropriate conditions for the reduction and oxidation of thepeptide.

The invention further provides an isolated nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary tosequence encoding a χ-conotoxin peptide as described above.

In a further aspect of the present invention there is provided a nucleicacid probe comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding all or part of a χ-conotoxinpeptide.

In a particularly preferred embodiment the nucleic acid probe comprisesa sequence of nucleotides encoding or complementary to a sequenceencoding the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein a reference to a “probe” includes reference to a primerused in amplification or a probe for use in direct hybridization.

Still another aspect of the present invention is directed to antibodiesto the χ-conotoxin peptides according to the invention. Such antibodiesmay be monoclonal or polyclonal and may be selected from naturallyoccurring antibodies to the peptides or may be specifically raised tothe peptides using standard techniques. In the case of the latter, thepeptides may first need to be associated with a carrier molecule. Theantibodies of the present invention are particularly useful astherapeutic or diagnostic agents.

In this regard, specific antibodies can be used to screen for thepeptides according to the invention. Techniques for such assays are wellknown in the art and include, for example, sandwich assays and ELISA.Knowledge of peptide levels may be important for monitoring certaintherapeutic protocols.

It may also be possible to prepare antiidiotypic antibodies usingtechniques known to the art. These antiidiotypic antibodies and theiruse as therapeutic agents represent a further aspect of the presentinvention.

The nucleic acid molecules of the present invention may be DNA or RNA.When the nucleic acid molecule is in DNA form, it may be genomic DNA orcDNA. RNA forms of the nucleic acid molecules of the present inventionare generally mRNA.

Although the nucleic acid molecules of the present invention aregenerally in isolated form, they may be integrated into or ligated to orotherwise fused or associated with other genetic molecules such asvector molecules and in particular expression vector molecules. Vectorsand expression vectors are generally capable of replication and, ifapplicable, expression in one or both of a prokaryotic cell or aeukaryotic cell. Preferably, prokaryotic cells include E. coli, Bacillussp and Pseudomonas sp. Preferred eukaryotic cells include yeast, fungal,mammalian and insect cells.

Accordingly, another aspect of the present invention contemplates agenetic construct comprising a vector portion and a gene capable ofencoding a peptide according to the invention.

Preferably, the gene portion of the genetic construct is operably linkedto a promoter on the vector such that said promoter is capable ofdirecting expression of the gene portion in an appropriate cell.

The present invention extends to such genetic constructs and toprokaryotic or eukaryotic cells comprising same.

Chimeras of χ-conotoxins such as MrIA, with other conotoxins oradditionally with other peptides or proteins, can be made to engineerthe activity into other molecules, in some instances to produce a newmolecule with extra functionality. This would preferably be done usingthe segment or segments of the sequence of these peptides that containthe pharmacophore. Where the pharmacophore is discontinuous, thesegments making up the pharmacophore should be positioned in the newconstruct to allow binding to the receptor. Chimeras with otherconotoxins may include additional Cys residues and additional disulphidebonds.

It is common for conotoxin peptides within an activity class to have asimilar pattern of disulphide bonding, with peptide loops between therespective cysteine residues. For χ-MrIA and χ-MrIB disulphide bondslink the first and fourth, and the second and third cysteine residues.This pattern is different from the binding pattern observed forα-conotoxin peptides. Despite this difference chimeric derivatives maybe prepared by substituting a loop of a χ-conotoxin peptide with theloop comprising a sequence from another peptide, including α-conotoxin.

The invention also includes dimers, trimers, etc. of χ-conotoxinpeptides as well as χ-conotoxin peptides bound to other peptides.

Preferably the χ-conotoxin peptides according to the invention have 10to 30 amino acids, more preferably 11 to 20.

The complete gene sequence for the naturally occurring χ-conotoxinpeptides may be obtained using a combined 5′ RACE and 3′ RACE strategycoupled with cloning and DNA sequencing.

The χ-conotoxin peptides according to the present invention are activein inhibiting neuronal noradrenaline transporter. Accordingly theinvention provides the use of a χ-conotoxin peptide as an inhibitor ofneuronal noradrenaline transporter, and in the treatment or prophylaxisof diseases or conditions in relation to which the inhibition ofneuronal noradrenaline transporter is associated with effectivetreatment. Such activity in pharmacological agents is associated withactivity in the prophylaxis or treatment of diseases or conditions ofthe urinary or cardiovascular systems, or mood disorders, or in thetreatment or control of pain or inflammation.

Accordingly the present invention provides a method for the treatment orprophylaxis of urinary or cardiovascular conditions or diseases or mooddisorders or for the treatment or control of pain or inflammationincluding the step of administering to a mammal an effective amount ofan isolated, synthetic or recombinant χ-conotoxin peptide having theability to inhibit neuronal noradrenaline transporter.

Examples of diseases or conditions of the urinary system include urinaryand fecal incontinence. Examples of cardiovascular diseases orconditions include arrhythmias of various origins and coronary heartfailure. Examples of mood disorders include depression, anxiety andcravings, such as smoking. Examples of pain include chronic pain,neuropathic pain and inflammatory pain.

Preferably the mammal is in need of such treatment although the peptidemay be administered in a prophylactic sense.

The invention also provides a composition comprising an isolated,synthetic or recombinant χ-conotoxin peptide having the ability toinhibit neuronal noradrenaline transporter, and a pharmaceuticallyacceptable carrier or diluent.

Preferably the composition is in the form of a pharmaceuticalcomposition.

There is also provided the use of an isolated, synthetic or recombinantχ-conotoxin peptide having the ability to inhibit neuronal noradrenalinetransporter in the manufacture of a medicament for the treatment orprophylaxis of urinary or cardiovascular conditions or diseases, or mooddisorders, of for the treatment or control of pain or inflammation.

It is also noted that noradrenaline transporter is expressed not only bynerve cells, but also by other tissues including the placenta, pulmonaryendothelial cells and the uterus. The peptides according to the presentinvention may also be effective in inhibiting these noradrenalinetransporters, and may be useful in treating conditions in which thesetransporters are implicated.

As will be readily appreciated by those skilled in the art, the route ofadministration and the nature of the pharmaceutically acceptable carrierwill depend on the nature of the condition and the mammal to be treated.It is believed that the choice of a particular carrier or deliverysystem, and route of administration could be readily determined by aperson skilled in the art. In the preparation of any formulationcontaining the peptide actives care should be taken to ensure that theactivity of the peptide is not destroyed in the process and that thepeptide is able to reach its site of action without being destroyed. Insome circumstances it may be necessary to protect the peptide by meansknown in the art, such as, for example, micro encapsulation. Similarlythe route of administration chosen should be such that the peptidereaches its site of action.

The pharmaceutical forms suitable for injectable use include sterileinjectable solutions or dispersions, and sterile powders for theextemporaneous preparation of sterile injectable solutions. They shouldbe stable under the conditions of manufacture and storage and may bepreserved against oxidation and the contaminating action ofmicroorganisms such as bacteria or fungi.

Those skilled in the art may readily determine appropriate formulationsfor the peptides or modified peptides of the present invention usingconventional approaches. Identification of preferred pH ranges andsuitable excipients, for example antioxidants, is routine in the art(see for example Cleland et al, 1993). Buffer systems are routinely usedto provide pH values of a desired range and include carboxylic acidbuffers for example acetate, citrate, lactate and succinate. A varietyof antioxidants are available for such formulations including phenoliccompounds such as BHT or vitamin E, reducing agents such as methionineor sulphite, and metal chelators such as EDTA.

The solvent or dispersion medium for the injectable solution ordispersion may contain any of the conventional solvent or carriersystems for peptide actives, and may contain, for example, water,ethanol, polyol (for example, glycerol, propylene glycol and liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about where necessary by the inclusion of various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal and the like. In many cases, it will bepreferable to include agents to adjust osmolality, for example, sugarsor sodium chloride. Preferably, the formulation for injection will beisotonic with blood. Prolonged absorption of the injectable compositionscan be brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.Pharmaceutical forms suitable for injectable use may be delivered by anyappropriate route including intravenous, intramuscular, intracerebral,intrathecal, epidural injection or infusion.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients such as these enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, preferredmethods of preparation are vacuum drying or freeze-drying a of apreviously sterile-filtered solution of the active ingredient plus anyadditional desired ingredients.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations preferably contain at least 1% by weight of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 5 to about 80%of the weight of the unit. The amount of active compound in suchtherapeutically useful compositions in such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such a sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

The present invention also extends to any other forms suitable foradministration, for example topical application such as creams, lotionsand gels, or compositions suitable for inhalation or intranasaldelivery, for example solutions or dry powders.

Parenteral dosage forms are preferred, including those suitable forintravenous, intrathecal, intracerebral or epidural delivery.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. A unit dosageform can, for example, contain the principal active compound in amountsranging from 0.25 μg to about 2000 mg. Expressed in proportions, theactive compound is generally present in from about 0.25 μg to about 200mg/ml of carrier. In the case of compositions containing supplementaryactive ingredients, the dosages are determined by reference to the usualdose and manner of administration of the said ingredients.

The invention will now be described with reference to the accompanyingdrawings and examples, however it is to be understood that theparticularity of the following description is not to supersede thegenerality of the preceding description of the invention.

Referring to the figures:

FIG. 1 is a graphical representation showing the typical effect ofχ-MrIA on the time course of the contraction of the bisected ratprostatic vas deferens to field stimulation with a single supramaximalpulse (55 V, 1 ms). χ-MrIA (30 nM-3 μM) was added to the organ bathcumulatively in half log unit steps.

FIG. 2 is a graphical representation of the effect of χ-MrIA, -MrIA oncontractile responses of bisected portions of the rat epididymal vasdeferens to exogenous α₁-adrenoceptor agonists. (a) logconcentration-response curves to noradrenaline in the absence (◯) andthe presence of 1 μM (Δ) or 3 μM (□) in χ-MrIA, -MrIA. (b) logconcentration-response curves to methoxamine in the absence (◯) and thepresence (□) of 3 μM χ-MrIA. Each data point in (a) and (b) representsthe mean±SEM of observations from 4-5 individual tissue preparations.Some error bars are obscured by the symbols.

FIG. 3 is a graphical representation showing inhibition by χ-MrIA on thedesipramine sensitive accumulation of [³H]-noradrenaline by ChineseHamster Ovary (CHO) cells transfected with the cDNA clone for the humanneuronal noradrenaline transporter. Accumulation of [³H]-noradrenalineis expression as a percentage of the cellular uptake in the absence ofχ-MrIA. Data points represent the mean±SEM or observations from 4separate experiments.

FIG. 4 is a diagrammatic representation showing derivation of coneshellvenom peptide sequences. 5′RACE PCR using the primers AP1+CHI-1B producethe 5′ UTR and leader peptide sequence which is then used to generatethe PCR primers specific for χ-conotoxins. The 3′ UTR using the primersCHI-1A+ANCHOR completed the derivation of the remaining mature peptidesequence and 3′ UTR sequence.

EXAMPLES

Drugs

The drugs used in this and the following examples include: desipraminehydrochloride (Sigma); indomethacin (Sigma); methoxamine hydrochloride(Sigma); (−)-noradrenaline bitartrate (Sigma); [³H]-1-noradrenaline(specific activity 2200 Ci/mM; New England Nuclear, Boston, Mass.,U.S.A.); tetrodotoxin (Sigma); yohimbine hydrochloride (Sigma).

Statistical Analysis

Data for the examples below are expressed as the mean and standard errorof 4-6 experiments. Sigmoidal curve-fitting for the calculation of EC₅₀values was performed by non-linear regression using the software packageIgor Pro (WaveMetrics). Differences between means were assessed byStudent's t test (two-tailed) using the software package Prism(GraphPad). Values of P<0.05 were taken to indicate statisticallysignificant differences.

Example 1

Rat Vas Deferens Preparation

Male Wistar rats (250-350 g) were killed by a blow to the head and thevasa deferentia were removed. Each tissue was divided into bisectedepididymal and prostatic portions. The tissue segments were mounted in 5mL organ baths under a tension of 0.5 g. The bathing solution had thefollowing composition (mM): NaCl, 119; KCl, 4.7; MgSO₄, 1.17; KH₂PO₄,1.18; NaHCO₃, 25.0; glucose, 5.5; CaCl₂, 2.5; EDTA, 0.026; wasmaintained at 37° C. and bubbled with 5% v/v. CO₂ in O₂. Thepreparations were equilibrated for at least 45 min prior to thecommencement of experimentation. Contractions were registered by meansof an isometric force transducer (Narco Bio-System F-60) and wererecorded on a Power Macintosh computer using Chart version 3.5.4/ssoftware and a MacLab/8s data acquisition system (ADInstruments) at asampling frequency of 200 Hz.

The bisected prostatic segments were used to examine the effect ofχ-MrIA on the electrically evoked contraction of the smooth musclemediated by sympathetic neurotransmission. The tissue preparations werestraddled with platinum stimulating electrodes. Electrical fieldstimulation (EFS) was made at 3 min intervals using a single 55 V pulseof 1 ms duration generated by a Grass S44 Stimulator. The contractionscould be blocked by tetrodotoxin (0.1 μM), indicating they were neurallymediated. Increasing concentrations of the peptide were added to theorgan bath cumulatively in half log unit increments. Each dose wasapplied once the effect of the previous dose on the response toelectrical stimulation had attained a steady level.

Effect of χ-MrIA on Sympathetic Neurotransmission

The bisected portions of the prostatic rat vas deferens responded tofield stimulation with a biphasic contraction. In this preparation, thetwo components of the biphasic contraction were well separatedtemporally. The first part of the response was the dominant one, andreached a maximum level approximately 200 ms after delivery of theelectrical pulse. The second phase of the contraction peakedapproximately 500 ms after stimulation. Our attempts to identify thepharmacological activity of χ-MrIA began with an investigation of itseffects in the field stimulated rat vas deferens. The effect of χ-MrIAon the response of the preparation to field stimulation is shown inFIG. 1. The conotoxin (30 nM-3 μM) acted to increase the second phase ofthe contraction. This effect was found to be concentration dependent. Bysubtracting the control response from traces obtained in the presence ofχ-MrIA, the specific enhancement by the peptide of only the secondcomponent of the contraction becomes apparent. A concentration-responsecurve for χ-MrIA acting to specifically potentiate the second componentcan also be constructed.

The action of χ-MrIA on the electrically evoked response was highlyspecific, enhancing only the second component of the biphasic response.This late phase of the contraction is recognised to be mediated bynoradrenaline, and is selectively inhibited by prazosin and otherα₁-adrenoceptor antagonists. The first phase of the contraction, whichis due to the activation of postjunctional P_(2x)-purinoceptors byreleased ATP, was not similarly enhanced. The magnitude of thenoradrenergic component of the contraction is modulated by the amount ofnoradrenaline released by sympathetic nerve firing, the density ofpostjunctional α₁-adrenoceptors and their coupling to effector systems,and the rate at which noradrenaline is cleared from the synapse.

Antagonism at presynaptic α₂-adrenoceptors is well recognised to enhancethe electrically evoked release of noradrenaline from sympathetic nervesby blocking the activation of a negative feedback system by releasednoradrenaline. However, α₂-adrenoceptor antagonism can not be themechanism of action of χ-MrIA. Unlike χ-MrIA, α₂-adrenoceptorantagonists such as yohimbine and idazoxan act to enhance equally thepurinergic and noradrenergic components of the contraction of the ratvas deferens. Furthermore, the response to a single pulse, as opposed toa train of stimuli, is not subject to regulation by this negativefeedback mechanism since there would be no agonist present at thesereceptor sites at the time of stimulation. Accordingly yohimbine (1 μM)has no effect on the evoked responses in this assay.

Example 2

Preparation to Examine Effect of χ-MrIA on Responses to α₁-AdrenoceptorAgonists

The rat vas deferens was used as described above, except that thebisected epididymal segments were not electrically stimulated. Thesepreparations were instead used to establish concentration responsecurves to noradrenaline and methoxamine in the absence and presence ofχ-MrIA. In this tissue, noradrenaline and methoxamine cause contractionof the smooth muscle via activation of postjunctional α₁-adrenoceptors.χ-MrIA at a concentration of either 1 μM or 3 μM was applied to theorgan bath and allowed to equilibrate with the tissue for 20 min priorto cumulative additions of noradrenaline or methoxamine. A singleconcentration response curve was determined per preparation, withcontralateral tissue segments to which χ-MrIA was not applied serving ascontrols.

Effect of χ-MrIA on Responses to α₁-Adrenoceptor Agonists

It was possible to determine whether the action of χ-MrIA occursupstream or downstream of neurotransmitter release by examining theeffect of the peptide on the response to exogenously appliednoradrenaline. Since χ-MrIA enhanced the potency of bath-appliednoradrenaline, we can conclude that the conotoxin acts by potentiatingthe response to noradrenaline, rather than by promoting its release fromneuronal stores. This potentiation could occur as a consequence ofincreased α₁-adrenoceptor responsiveness or impaired termination of theaction of noradrenaline. The α₁-adrenoceptor agonist methoxamine wasused to ascertain which of these was the mechanism of action of χ-MrIA.This α₁-adrenoceptor agonist differs from noradrenaline in that it isnot a substrate for the neuronal noradrenaline transporter. Thistransporter functions to eliminate noradrenaline and othercatecholamines from the synapse by uptake into the nerve terminals, andrepresents the major mechanism for terminating the action ofnoradrenaline at the adrenoceptors of sympathetically innervatedtissues. Because methoxamine is not subject to removal by thismechanism, inhibition of the transporter does not enhance the potency ofits actions.

The effect of χ-MrIA on the responses of the bisected segments of therat epididymal vas deferens to two α₁-adrenoceptor agonists wasinvestigated. Log concentration-response curves to noradrenaline in theabsence and presence of χ-MrIA are shown in FIG. 2 a. At a concentrationof 1 μM, χ-MrIA acted to increase the sensitivity of the tissue tonoradrenaline, shifting the concentration response curve to the left.The degree of potentiation observed was larger in experiments with 3 μMχ-MrIA. Neither concentration of the conotoxin altered the maximumresponse of the tissue to noradrenaline. χ-MrIA at a concentration of 3μM had no effect on the concentration response curve to methoxamine(FIG. 2 b). The observation that χ-MrIA does not potentiate the actionof methoxamine, in contrast to its effect on responsiveness tonoradrenaline, is consistent with χ-MrIA being an inhibitor of theneuronal noradrenaline transporter.

The lack of effect of χ-MrIA on the concentration response curve tomethoxamine also demonstrates that χ-MrIA has no effect atα₁-adrenoceptors, which would be evident as a parallel shift of thecurve to the right. This distinguishes χ-MrIA from some other inhibitorsof noradrenaline transport, particularly those used as antidepressants.The therapeutic target of many of the antidepressant drugs is theneuronal noradrenaline transporter. However, many of these compounds,especially the tricyclic antidepressants, and to a lesser extent somenewer drugs which are structurally unrelated to the conventionaltricyclics, are recognised to act at other sites such asα₁-adrenoceptors and muscarinic ACh receptors.

Example 3

Guinea-Pig Ileum

Male guinea-pigs (285-425 g) starved overnight were killed by a blow tothe head and exsanguinated. Segments of the ileum of approximately 1.5cm length were removed and the luminal contents cleared by gentlewashing using a syringe filled with bathing solution. The preparationswere placed in 5 mL organ baths containing bathing solution of thefollowing composition (mM): NaCl, 136.9; KCl, 2.68; CaCl₂, 1.84; MgCl₂,1.03; glucose, 5.55; NaHCO₃, 11.9; and KH₂PO₄, 0.45; warmed to 37° C.and bubbled with 5% v/v CO₂ in O₂. Indomethacin (10 μM) was included inthe bathing solution to produce a stable baseline. The tissues wereplaced under a tension of 1.0 g and allowed to equilibrate for 40 minprior to the commencement of experimentation. Doses of nicotine (4 μM)were then applied at 15 min intervals until the responses were observedto be consistent. χ-MrIA (3 μM) was then added, 25 min after whichanother dose of nicotine was applied. The responses to nicotine weremeasured isometrically and digitised at a sampling rate of 10 Hz.

Effect of χ-MrIA on Responses to Nicotine in the Guinea-Pig Ileum

χ-MrIA (3 μM) had no significant effect on the responses of ilealsegments to nicotine. In the absence of the conotoxin, the mean responsewas 3.83±0.76 g, compared to 4.07±0.80 g when χ-MrIA was present (p>0.1;paired t-test; n=4). The α-conotoxins block nicotinic ACh receptors ofeither the neuronal or muscle subtype. It was demonstrated that χ-MrIAdoes not target neuronal nicotinic ACh receptors using isolated segmentsof guinea-pig ileum. Because of the dependence of the contractileresponse on muscarinic receptor activation, it would be expected thatthe response of the guinea-pig ileum to nicotine would be attenuated inthe presence of χ-MrIA if the conotoxin also acted as a muscarinic AChreceptor antagonist. In this preparation, the nicotine-induced releaseof ACh and various other neurotransmitters which activate postjunctionalreceptors was unaffected by χ-MrIA. Thus, and in contrast to many othertransport inhibitors, χ-MrIA lacks anti-muscarinic activity.

Example 4

Mouse Phrenic Nerve-Hemidiaphragm Preparation

Male Quackenbush mice (20-30 g) were killed by cervical dislocation.Left and right hemidiaphragms were removed with the phrenic nervesattached. The base of each hemidiaphragm was positioned between twoparallel platinum stimulating electrodes and the phrenic nerve wasplaced through two small platinum loops for field stimulation. Thepreparation was incubated at 37° C. in a 5 mL organ bath bubbled with 5%v/v CO₂ in O₂. The composition of the bathing solution was (mM): NaCl,135.0; KCl, 5.0; CaCl₂, 2.0; MgCl, 1.0; glucose, 11.0; NaHCO₃, 15.0; andKH₂PO₄, 1.0. The tissues were placed under 1.0 g resting tension. Afterallowing at least 30 min for equilibration, alternating direct andindirect stimulation was made at 10 s intervals using a 30 V pulse of 2ms duration to the muscle, and a 3 V pulse of 0.2 ms duration to thenerve, respectively. The effect of a single dose of χ-MrIA at aconcentration of 3 μM on the directly and indirectly elicitedcontractions was examined. Responses were digitised and recorded asdescribed for the vas deferens preparations.

Effect of χMrIA on Responses to Electrical Stimulation of the MousePhrenic Nerve-Hemidiaphragm

Contractions elicited by field stimulation of the phrenic nerve, or bydirect muscle stimulation, were unaffected by 3 μM χ-MrIA (n=4). χ-MrIAdoes not block the muscle nicotinic ACh receptors in the mousephrenic-nerve hemidiaphragm preparation. The lack of activity of χ-MrIAat skeletal muscle or motor nerve distinguishes it from the majority ofconotoxin peptides characterised to date which are paralytic toxins andhence have a clear role in prey capture.

Example 5

Cellular Uptake of [³H]-Noradrenaline

Chinese hamster ovary (CHO) cells were grown in 24 well plates (Falcon)in 10% v/v fetal calf serum. On reaching 60-70% confluence, the cellswere transiently transfected (Lipofectamine, Gibco) with an expressionvector (pcDNA3, Invitrogen) incorporating the full length cDNA for thehuman neuronal noradrenaline transporter (Pacholczyk et al., (1991)Nature, 350, 350-4). A cDNA clone of the neuronal noradrenalinetransporter was used (Vollum Institute, Portland, Oreg., USA). Cellularuptake studies were performed 36 hours after transfection. The CHO cellswere initially washed with transport buffer containing (mM): NaCl, 157;KCl, 2.7; NaH₂PO₄, 11.8; MgCl₂, 1.0 and CaCl₂, 0.1; and of pH 7.4. Thecells were then incubated with transport buffer containing 50 nM[³H]-noradrenaline (supplemented with unlabelled noradrenaline asrequired) and 100 μM ascorbic acid. χ-MrIA (0.1 nM-1 μM) or desipramine(10 μM) were also included as appropriate. After 20 minutes at roomtemperature, the cells were rapidly washed with ice-cold phosphatebuffered saline and then lysed in 0.1% v/v Triton-X. The cell lysateswere taken for liquid scintillation counting to determine their level ofradioactivity. Additionally, an aliquot of the cell lysate was used tomeasure protein concentration (BioRad DC protein assay). The specificuptake of [³H]-noradrenaline by the noradrenaline transporter wasdefined as the component sensitive to desipramine (10 μM).

Effect of χ-MrIA on Cellular Accumulation of [³H]-Noradrenaline

The accumulation of noradrenaline into CHO cells expressing the humanneuronal noradrenaline transporter was reduced to less than 0.5% of thecontrol amount by desipramine (10 μM), demonstrating that theaccumulation was due almost entirely to specific uptake via the clonedtransporter. The noradrenaline transporter was confirmed as the targetof the conotoxin in cellular uptake studies. χ-MrIA (0.1 nM-1 μM)inhibited the accumulation of radiolabelled noradrenaline in aconcentration-dependent manner (FIG. 3), with a log IC₅₀ value of−8.17±0.0275 (n=4). The concentration of χ-MrIA required to inhibit theaccumulation by 50% was found to be approximately 7 nM. Thisconcentration is approximately one order of magnitude lower than thatneeded for desipramine to produce the same effect.

Cocaine and χ-MrIA are both naturally occurring compounds, however, theyare quite dissimilar. Cocaine is an alkaloid extracted from the leavesof the coca plant, whereas χ-MrIA is a peptide directly encoded by ananimal gene. In addition to its effect at the uptake transporter,cocaine is known to possess potent local anaesthetic properties. This isdue to blockade of both sodium and potassium channels. No evidence wasfound for local anaesthetic activity of χ-MrIA in any of the assays. Itwas found that χ-MrIA had neither contractile nor relaxant effects onthe tone of the vas deferens by itself. Similar studies revealed thatα-conotoxin does not inhibit the dopamine transporter.

Example 6

Tritiated mazindol binding to the noradrenaline transporter was measuredin cells expressing the transporter protein (see Example 5). Theinfluence of χ-MrIA from 10⁻⁶ to 10⁻⁹ on tritiated mazindol binding wasmeasured. χ-MrIA had no effect on tritiated maizindol binding,indicating that it acts non-competitively, at a site distinct fromtraditional noradrenaline transport inhibitors, such as desipramine,mazindol and cocaine.

Example 7

Derivation of Gene Sequence for the χ-Conotoxin Peptides

The complete gene sequence for the χ-MrIA was isolated using a combined5′ RACE (Random Amplification of cDNA Ends) and 3′ RACE strategy coupledwith cloning and DNA sequencing.

5′ RACE

The oligonucleotide primer CH1-1B were designed from the mature peptidesequence. The relationship of the oligonucleotides to the peptide is asfollows, together with the oligonucleotide sequence: χ-MrIA  -NGVCCGYKLCHPC SEQ ID NO. 3 CHI-1B 5′-CANGGRTGRCANARYTTRTA-3′ SEQ IDNO. 4 AP1 5′-CCATCCTAATACGACTCACTATAGGGC-3′ SEQ ID NO. 5(where N = A/C/G/T, R = A/G, Y = C/T,)

Polymerase Chain Reaction (PCR) was carried out using theoligonucleotide CH1-1B in combination with the AP1 oligonucleotide oncDNA templates derived from the mRNA isolated from coneshell venomducts. The PCR products, which represent the 5′ region of the MrIA genewere isolated, purified, cloned into bacterial vectors and sequenced.Gene sequence for MrIA was obtained from C. marmoreus (FIG. 4).

3′ RACE

The DNA sequence for the 5′-regions of the gene was used to designoligonucleotides that were capable of detecting the MrIA sequence, andsequences from other closely related peptides. The positioning of theoligonucleotides relative to the gene sequence is shown in FIG. 4. Theoligonucleotide CH1-1A was used in PCR in conjunction with the ANCHORoligonucleotide to produce DNA fragments corresponding to the leaderpeptide, mature peptide and 3′ untranslated (3′UTR) regions of the gene.PCR of venom duct cDNA templates from C. marmoreus produced DNAfragments corresponding to the MrIA peptide.

The DNA sequences for the oligonucleotides are: CHI-1A5′-ACAGGCAGAATGCGCTGTCTCCC-3′ SEQ ID NO. 6 ANCHOR5′-AACTGGAAGAATTCGCGGCCGCAGGAAT-3′ SEQ ID NO. 7Complete Sequence for χ-MrIA

Gene sequence for χ-MrIA produced using 5′ RACE and 3′ RACE representoverlapping fragments of the gene. These fragments were combined, toproduce a consensus sequence for the gene. The consensus sequence is thefull cDNA for the gene, and includes 5′ UTR, the leader peptide, themature peptide and the 3′ UTR. The χ-MrIA leader and mature peptideoligonucleotide sequence is shown in SEQ ID NO. 8, while the leader andmature peptide amino acid sequence is shown in SEQ ID NO. 9.ATGCGCTGTCTCCCAGTCTTGATCATTCTTCTGCTGC SEQ ID NO. 8TGACTGCATCTGCACCTGGCGTTGTTGTCCTACCGAAGACCGAAGATGATGTGCCCATGTCATCTGTCTACTGTAATGGAAAGAGTATCCTACGAGGAATTCTGAGGAACGGTGTGTGCTGTGGCTATAAGTTGTGCCATCCATGTTA AMRCLPVLIILLLLTASAPGVVVLPKTEDDVPMSSVYC SEQ ID NO. 9NGKSILRGILRNGVCCGYKLCHPC

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1-27. (canceled)
 28. An isolated, synthetic or recombinant χ-conotoxinpeptide having the activity of inhibiting a neuronal amine transporter,wherein said χ-conotoxin peptide comprises MrIA as set forth in SEQ IDNO: 1 (NGVCCGYKLCHOC) or such a sequence which has undergone one or moreamino acid deletions, additions, substitutions or sidechainmodifications, wherein “O” represents 4-hydroxyproline and wherein thefirst and fourth cysteine residues of SEQ ID NO: 1 are connected to forma disulphide or diselenium bond and the second and third cysteineresidues of SEQ ID NO: 1 are connected to form a disulphide ordiselenium bond.
 29. A χ-conotoxin peptide according to claim 28 havingthe ability to inhibit a neuronal noradrenaline transporter.
 30. Aχ-conotoxin peptide according to claim 29 which is a selective inhibitorof neuronal noradrenaline transporter.
 31. A χ-conotoxin peptideaccording to claim 29 having negligible or no anticholinergic effect.32. A χ-conotoxin peptide according to claim 29 having negligible or noactivity as a sodium channel blocker.
 33. A χ-conotoxin peptideaccording to claim 29 having negligible or no activity as an inhibitorof dopamine transporter.
 34. An isolated, synthetic or recombinantχ-conotoxin peptide having the activity of inhibiting a neuronal aminetransporter, wherein said χ-conotoxin peptide comprises MrIA as setforth in SEQ ID NO: 1 (NGVCCGYKLCHOC) or such a sequence which hasundergone an amino acid substitution or sidechain modification of theN-terminal residue and wherein “O” represents 4-hydroxyproline.
 35. Aχ-conotoxin peptide according to claim 34 having the ability to inhibita neuronal noradrenaline transporter.
 36. A χ-conotoxin peptideaccording to claim 35 which is a selective inhibitor of neuronalnoradrenaline transporter.
 37. A χ-conotoxin peptide according to claim35 having negligible or no anticholinergic effect.
 38. A χ-conotoxinpeptide according to claim 35 having negligible or no activity as asodium channel blocker.
 39. A χ-conotoxin peptide according to claim 35having negligible or no activity as an inhibitor of dopaminetransporter.
 40. A method for the treatment or control of pain in amammal comprising administering to the mammal an amount of a χ-conotoxinpeptide effective to treat or control pain, wherein said χ-conotoxinpeptide is a peptide as claimed in claim 28 or claim
 34. 41. Acomposition comprising a χ-conotoxin peptide and a pharmaceuticallyacceptable carrier or diluent, wherein said χ-conotoxin peptide is apeptide as claimed in claim 28 or claim
 34. 42. A composition accordingto claim 41 which is a pharmaceutical composition.